| Abstract |
The urinary bladder is a musculomembranous subperitoneal organ which functions as
a reservoir for the urine in the human body. Urinary bladder cancer is the 11th most
common malignancy worldwide and the 4th most common among males in the United
States, where 74,690 new cases will be diagnosed in 2014 alone. Smoking and
occupational exposure to aromatic amines are recognized as major risk factors for the
development of the disease, followed by chronic urinary tract infection, Schistosoma
infection in certain geographical regions, exposure to cyclophosphamide and
radiotherapy to adjacent organs. It is estimated that 90-95% of bladder cancer cases
are urothelial carcinomas, whereas the remaining 5-10% are mostly squamous cell
carcinomas and adenocarcinomas. Determination of tumor stage at diagnosis is crucial
for prognosis and patient management, as two major phenotypic variants of urothelial
tumors with drastically different biological behavior have been recognized. Low-grade
non-muscle-invasive tumors, comprising approximately 80% of the cases, have limited
invasive potential and come with favorable prognosis when treated early, although
accompanied by high recurrence rates. On the contrary, high-grade muscle-invasive
tumors have very bad prognosis, with most of the patients developing life-threatening
metastases despite surgical resection and systemic therapy. The genetic alterations
underlying the development of urothelial cancer include chromosomal aberrations,
inactivating tumor-suppressor gene mutations and activation of oncogenes. FGFR3
mutations are detected in more than 70% of non-muscle-invasive tumors, whereas p53
is mutated in almost 50% of the muscle-invasive lesions.
TP53 is the most extensively studied tumor-suppressor gene. Mutations in the TP53
gene have been reported in the majority of human malignancies, suggesting a
significant contribution to the development and progression of human tumors. Two
TP53 homologs, TP63 and TP73, have recently been identified. Though highly similar
with p53 regarding genomic organization, both p63 and p73 utilize complex
mechanisms to provide two major classes of protein isoforms with tumor-suppresive
(TA-isoforms) or tumorigenic function (ΔN-isoforms). TAp63 and TAp73 exhibit p53-like
function in terms of inducing apoptosis, whereas the N-terminally truncated ΔNp63
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and ΔNp73 act as dominant negative inhibitors of both wild-type p53 protein and the
respective TA-isoforms. Both TP63 and TP73 are rarely mutated in human tumors,
whereas their deletion from mice results in various developmental and neurological
abnormalities rather than the development of malignant lesions. Overall, it is evident
that the two TP53 homologs are not to be considered as classic tumor-suppressor
genes.
The objective of this PhD thesis was to determine the expression levels of the three
p53 family genes (TP53, TP63, TP73) and the TP63 and TP73 TA and ΔN isoforms in
bladder tumors and to evaluate any alterations in their expression patterns that might
be associated with the development and progression of urothelial malignancies. The
expression analysis was performed in a set of 30 urothelial tumors, matched with
normal tissues, which were obtained from bladder cancer patients who underwent
surgical resection of bladder tumor.
Expression analysis revealed elevated p53, p63 and p73 mRNA levels in 47.0%, 40.0%
and 43.0% of the samples, respectively. p53, p63 and p73 exhibited a 1.9-fold, 3.0-fold
and 2.0-fold overexpression in tumor specimens compared with the normal samples,
respectively. In the normal bladder epithelium, all members of the p53 gene family
were co-expressed (p΄&λτ0.001). However, in malignant urothelium, only the positive coexpression
between p63 and p73 was maintained (p=0.008). Statistical analysis
revealed that p53 was up-regulated in non-muscle-invasive (pTa-T1) tumors compared
with muscle-invasive (pT2-T4) ones (p=0.047). Significantly higher p63 mRNA levels
were measured in malignant samples from patients ΄&γτ 75 years of age versus younger
patients (p=0.022) and from patients with disease relapse compared with cancer-free
patients (p=0.045). p63 expression significantly deviated from the normal pattern in
papillary versus non-papillary lesions (p=0.026). p73 overexpression was observed in
Grade III tumors compared with Grade II tumors, in patients presenting with one-site
lesions (p=0.040).
Also, elevated TAp63, ΔNp63, TAp73 and ΔNp73 mRNA levels were measured in 33.0%,
50.0%, 40.0% and 37.0% of the samples, respectively. TAp63, ΔNp63, TAp73 and
ΔNp73 exhibited a 2.9-fold, 3.2-fold, 1.7-fold and 3.3-fold overexpression in tumor
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specimens compared with the normal samples, respectively. Tumor-specific TAp63
expression was detected in 3 cases, while TAp63 was expressed only in normal tissue
in 4 cases. Tumor-specific ΔNp73 expression was detected in 7 cases, while ΔNp73 was
expressed only in normal tissue in 4 cases. ΔNp73 was also not expressed in 3 tissue
pairs. The ΔNp63/TAp63 isoform ratio shifted 1.1-fold in favor of ΔΝp63, and the
ΔNp73/TAp73 isoform ratio shifted 2.0-fold in favor of ΔΝp73 from normal to tumor
specimens. In normal urothelium TAp73 was co-expressed with ΔNp73 (p=0.003). In
the malignant bladder epithelium ΔNp63 negatively correlated with TAp73 (p=0.006).
Statistical analysis revealed that ΔΝp63 was up-regulated in tumor samples from
bladder cancer patients ΄&γτ 75 years old versus younger ones (p=0.026), in non-muscleinvasive
(pTa-T1) tumors versus muscle-invasive ones (pT2-T4) (p=0.019) and in
patients who received BCG treatment versus those who were treated with cystectomy
(p=0.045). TAp73 mRNA levels were increased in patients with papillary tumors
compared with those with non-papillary ones (p=0.006) and in current smokers
compared with non-smokers and ex-smokers combined (p=0.020).
Our results indicate that expression of all TP53 family members deviate from the
normal pattern in bladder tumors. p63 overexpression is associated with a less
aggressive tumor phenotype, whereas p73 up-regulation correlates with an
unfavorable patient outcome. We also provide evidence that p63 and p73 exert their
contribution to the malignant transformation of the bladder epithelium independently
of p53. In addition, our results support an anti-invasive role for ΔNp63 in urothelial
cancers and a potentially protective effect of TAp73 on bladder epithelium. Finally, we
provide evidence for epigenetic regulation of p63 and p73 isoforms expression in
normal and malignant urothelium.
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Expression analysis of the TP53,TP63 and TP73 genes and the TA p63,ΔΝρ63,ΤΑρ73 και ΔΝρ73 isoforms in urinary bladder cancer
